The controlled organization of inorganic materials into multi-dimensional addressable arrays is the foundation for both logic and memory devices, as well as other nonlinear optical and sensing devices (Zhirnov et al., 2001 Computer 34: 34-43; Xia et al., 2000 Adv. Mater. 12: 693-713). Many of these devices are currently fabricated using lithographic patterning processes that have progressively developed toward greater integration densities and smaller sizes. At submicron scales, however, conventional lithographic processes are approaching their practical and theoretical limits. At scales below 100 nm, ion and electron beam lithography becomes prohibitively expensive and time consuming, and more importantly, at these scales quantum effects fundamentally change the properties of devices (Sato et al., 1997 J. Appl. Phys. 82: 696).
Nanoscale templates for constrained synthesis, in situ deposition, or direct patterning of nanometer scale inorganic arrays are being developed using both artificial and natural materials. Artificial materials such as microphase separated block copolymers (Park et al., 2001 Appl. Phys. Lett. 79: 257-259) and hexagonally close-packed spheres (Hulteen et al., 1995 J. Vac. Sci. Technol. A, 1553-1558) have been used for nanoscale fabrication. Natural materials such as DNA (Richter et al., 2000 Adv. Mater. 12: 507-510; Keren et al., 2002 Science 297: 72-75), bacterial and archaeal surface layer proteins (S-layer proteins) (Sleytr et al., 1999 Angew. Chem. Int. Ed. 38: 1034-1054; Douglas et al., Appl. Phys. Lett. 48: 676-678; Hall et al., 2001 CHEMPHYSCHEM 3: 184-186), virus capsids (Shenton et al., 1999 Adv. Mater. 11: 253-256; Douglas et al., 1999 Adv. Mater., 679-681; Douglas et al., Nature 393: 152-155; Wang et al., 2002 Angew. Chem. Int. Ed. 41: 459-462), phage (Lee et al., 2002 Science 296: 892-895), and some globular proteins (Yamashita, I., 2001 Thin Solid Films 393: 12-18) have been used as templates and in other nanoscale applications.
Various nanometer scale objects, including arrays of nanoparticles formed by non-conventional methods are being explored for use as viable alternatives to standard lithographically patterned devices. Individual nanoparticles, also known as quantum dots (QDs), have been shown to behave as isolated device components such as single electron transistors (Likharev, K. K., 1999 Proc. IEEE 87: 606-632; Thelander et al., 2001 Appl. Phys. Lett. 79: 2106-2108). Theoreticians have postulated that two-dimensional arrays of QDs with nanoscale resolution could form the basis of future generations of electronic and photonic devices. The function of these devices will be based on phenomena such as coulomb charging, inter-dot quantum tunneling and other coherent properties derived from the electronic consequences of confinement and nanoparticle surface area to volume ratios (Maier, S. A. et al., 2001 Adv. Mater. 13: 1501-1505; Maier et al., Phys. Rev. B 65, 193408; Zrenner, A. et al., 2002 Nature 418: 612-614; Berven et al., 2001 Adv. Mater. 13: 109-113).
Traditional techniques for patterning ordered arrays of materials onto inorganic substrates and manufacturing devices currently used are ion beam lithography and molecular beam epitaxy. These techniques possess inherent limitations due to the use of polymeric light masks for pattern formation, however, there is a theoretical limitation of patterning that could ultimately limit the processes in the hundreds of nanometers.
While there are strong incentives to develop nanoscale architectures, these developments require alternate fabrication methods and new insights into the behavior of materials on nanometer scales (Nalwa, H. S., 2000 “Handbook of Materials and Nanotechnology”, Academic Press, San Diego).
Development of methods for ordering nanoscale materials through “bottom up” assembly will provide new tools for creating nanostructured materials and devices that self-assemble or self-repair. Synthetic and biological polymers have gained attention because of their inherent ability to form structures on the nanometer scale with little or no mechanical processing. Self-assembly and phase separation of these natural or synthetic polymers have been successfully used for nanoscale ordering of materials. Biopolymers form especially well-defined structures and assemblies with highly specific chemical functionalities. Nucleic acids (J Richter, et al., 2000 Advanced Materials 12:507-510; M G Warner and J E Hutchison 2003 Nature Materials 2:272-277; and K Keren, et al., 202 Science 297:72-75), proteins (K Douglas and N A Clark 1986 Appl Phys Lett 48:676-678; U B Sleytr, et al., 1999 Angew Chem Int Edn 38:1034-1054; I Yamashita 2001 Thin Solid Films 393:12-18; M Allen, et al., 2002 14:1562-1565; R A McMillan, et al., 2002 Nature Materials 1:247-252), virions and virus capsids (W Shenton, et al., 1999 Adv Mater 11:253-256; S-W Lee, et al., 2002 Science 296:892-895; Q Wang, et al., 2002 Angew Chem Int Ed Engl 41:459-462) have all been used to create nanostructured materials with unique properties.
A number of protein complexes have been developed as nanoscale templates. These templates can be functionalized by genetic modification to add chemically reactive sites that bind inorganic materials. For example, chaperonin complexes can be functionalized to bind soft metals. In nature, chaperonins are protein complexes having two stacked rings each comprising 7, 8 or 9 HSP60 subunits. The HSP60 subunits were mutated to include single cysteine residues placed at different solvent-exposed sites, including the apical pore. The thiols of these cysteine residues provide binding sites for gold or zinc (PCT/US02/35889). The chaperonin complexes comprising these mutant HSP60 subunits bind gold or zinc and assemble into two-dimensional crystals.
Protein complexes can also be modified to include peptide sequences having desirable binding or catalytic functions. These protein complexes comprise subunits having inserted peptide sequences. However, the mutant subunits may fail to fold, assemble into complexes or organize into higher-order structures. Furthermore, insertion as a loop may render the peptide sequence inactive and fusion to one of the native termini may not provide sufficient surface accessibility. To overcome this challenge, circular permutation has been used to join peptide sequences within a protein template. Circular permutation is a reordering of the polypeptide chain such that the original N- and C-terminal ends are joined and new termini are created elsewhere in the protein. New peptide sequences can be joined to either of the new termini without perturbing subunit assembly. Published studies of protein circular permutation demonstrate that, for proteins in which the native amino and carboxyl termini are near in space, many relocated positions for the new termini are viable (P T Beernink, et al., 2001 Protein Sci 10:528-537; U Heinemann and M Hahn 1995 Prog Biophys Mol Biol 64:121-143; M Iwakura, et al., 2000 Nat Struct Biol 7:580-585).
The present invention provides chaperonin subunit polypeptides which are modified to relocate the native N-terminal and C-terminal ends from the central pore region to various new positions on the exterior of the folded modified chaperonin polypeptide. The relocated N- and C-terminal ends are joined with a peptide sequence that behaves as a reporter. The modified chaperonin polypeptides fold into subunits that self-assemble into double-ringed chaperonin structures, and the chaperonin structures organize into higher order structures such as two-dimensional crystals and filaments. Additionally, the reporter peptide is functional. These chaperonin structures are useful for producing ordered nanoscale materials and devices.